Mitochondrial NHE1 and NCLX in the diabetic cardiomyopathy

RAYEN DE FAZIO; FERNANDA ELIZABETH CARRIZO VELASCO ; NOELIA COSTANTINI ; SOFÍA CIARROCCHI; ERNESTO ALEJANDRO AIELLO; CAROLINA JAQUENOD DE GIUSTI

Congreso; SCHCF + ALACF 2020 joint meeting; 2020

(1) Advanced Center for Chronic Diseases (ACCDiS), Faculty of Chemical and Pharmaceutical Sciences & Faculty of Medicine,University of Chile, Santiago, Chile.(2) Department of Internal Medicine (Cardiology Division), University of Texas Southwestern Medical Center, USA.Introduction: The classic renin-angiotensin system (RAS) has a counter-regulatory axis known as alternative RAS withcardioprotective action on the properties of cardiomyocytes. Angiotensin-(1-9) (Ang-(1-9)) blocked mitochondrial fission in amodel of norepinephrine-induced cardiomyocyte hypertrophy. This latter cellular process is characterized by increases in the sizeof cardiomyocytes, the number of sarcomeres, the content of contractile proteins (i.e., the beta-myosin heavy chain), and by there-expression of the fetal gene program (i.e. ANF). Moreover, mitochondria are fragmented in this process, thus decreasingoxidative metabolism. Interestingly, excessive accumulation of lipids (lipotoxicity) in the heart also promotes morphological andmetabolic changes in cardiomyocytes, including the development of cardiac hypertrophy. Aim: To evaluate if Ang-(1-9) preventsthe effects of lipotoxicity on cardiac hypertrophy. Methodology: Neonatal rat ventricular myocytes (NRVM) were isolated fromnewborn (2-3 days) Sprague-Dawley rat hearts in a procedure approved by the institutional committee for the care and use ofanimals of the University of Chile. NRVMs were treated with or without 100 μM Ang-(1-9) for 6 h before treatment with palmitate(328 μM for 24 h) and then, hypertrophic changes were evaluated. Different hypertrophic markers were assessed, including β-MHC and ANP protein levels by Western blot. Cell area, perimeter, and mitochondria mean volume and number were analyzedby confocal microscopy. Results are expressed as mean ± SEM. Statistical analysis corresponds to the T-test or one-way Anovaanalyses of 2-4 independent experiments (N). Results: Palmitate (328 μM 24 h) significantly increases the levels of β-MHC andANP, increases the area and perimeter of the NVRMs and triggered mitochondrial fragmentation. Ang-(1-9) prevents the increaseof β-MHC, suggesting that it could prevent cardiomyocyte hypertrophy and the changes in mitochondrial morphology induced bylipotoxicity. Conclusions: Palmitate promotes cardiomyocytes hypertrophy together with changes in mitochondrial morphology.Ang-(1-9) prevents lipotoxicity-induced cardiomyocyte hypertrophy.Funding: FONDECYT 1161156 (SL), 1190743 (VP), FONDAP 15130011 (SL, VP), CRP-ICGEB CHL18-04 (VP) and CONICYT PhDFellowship 21181428 (FM).KD851GGNeuronal activity modulates pericyte interactions via pannexons: characterization, mechanisms and implicationsJuan Irigoyen1, Sandra Mai1, Eugenia Isasi2, Verónica Abudara1(1) Universidad de la República, Fisiología, Facultad de Medicina, General Flores 2125, Montevideo 11 800, Uruguay.(2) Universidad de la República, Histología y Embriología, Facultad de Medicina, General Flores 2125, Montevideo 11 800, Uruguay.Introduction: To avoid energy deprivation, the cerebral local blood flow (BF) increases in active areas to adjust glucose and oxygensupply to neurometabolic demands, a response known as functional hyperemia. The Neurovascular Unit coordinates this process;however, its intimate mechanisms and regulation have not been resolved. In the neuro-microvascular interface, the contractilepericapillary pericytes, sense the neuronal activity and undergo relaxation increasing the BF. Brain pericytes express large-porechannels formed by pannexin1 (pannexons); open pannexons release ATP, increasing intracellular calcium. Objectives:Characterize the role and regulation of Pannexin1 in cerebral pericapillary pericytes during increased neuro-metabolic demands.Methods: Using pharmacological tools, functional imaging and molecular biology, we evaluated pannexons activity inhippocamapal pericytes of wild-type and pannexin1 knock-out (Panx1-/-) mice in resting conditions and upon increased neuronaldischarge, in both ex vivo and in vivo conditions. We employed Mann-Whitney and Kruskal-Wallis tests for statistical analysis. TheAnimal Experimentation Committee of Universidad de la República, Montevideo-Uruguay, approved all animal procedures.Results: Epileptic seizures (in vivo) induced by picrotoxin-PTX (1mg/kg; 8mg/kg ip), a GABA-A receptor blocker, significantlyinhibited hippocampal pericyte membrane permeability as compared to control (vehicle). This effect was confirmed ex vivo inPTX-treated (100μM; 45min) acute hippocampal slices. Genetic ablation of pannexin1 (Panx1-/- mice), or administration oftetrodotoxin (TTX; 0.5μM), an inhibitor of voltage-gated sodium channels or DPCPX (5μM; 10μM), an A1 adenosine receptorantagonist, but not MRS1754 and ANR-94, respectively A2a and A2b adenosine receptor antagonists, totally prevented PTX-inhibition. Adenosine mimicked PTX effects in slices and cultured pericytes. Conclusion: Our results indicate that neuronal activitydecreases the molecular exchange between brain pericytes and their microenvironment under both ex vivo (slices) and in vivo(awake animal) conditions; this effect is due to the closure of pannexons and could be mediated by adenosine through A1receptors, probably via a direct effect.Financiado por la Comisión Sectorial de Investigación Científica y la Agencia Nacional de Investigación e Innovación - Udelar(Uruguay)TQ973QCMitochondrial NHE1 and NCLX in the diabetic cardiomyopathyRayen De Fazio1, Fernanda Elizabeth Carrizo Velasquez1, Noelia Costantini1, Sofía Ciarrocchi1, Ernesto Alejandro Aiello1, CarolinaJaquenod De Giusti1(1) Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Médicas, UNLP-CONICET, La Plata, Argentina.Introduction: Heart failure is the leading cause of death among diabetic people. Cellular and molecular entities leading to diabeticcardiomyopathy (DCM) are poorly understood. Na+/H+ exchanger (NHE) is a ubiquitous ion exchanger responsible for intracellularVIDEO POSTER ABSTRACTSMolecular and Cellular Physiology63pH maintenance. NHE1 is the heart isoform. Our previous results show increased NHE1 activity in the heart from obese anddiabetic mice (ob-/-) compared to the control heterozygous littermates (ob+/-). Mitochondrial dysfunction has been related tothe development of heart failure and NHE1 has been detected in rat mitochondria, where its inhibition resulted in decreasedmitochondrial swelling. Altered mitochondrial Ca2+ may result in mitochondrial dysfunction. The role of mitochondrial NHE1 andNa+/Ca2+ exchanger (NLCX) in DCM has not been yet studied. Objective to study the role of NHE1 and NLCX in the mitochondriaof ob-/- mice. Methodology: Left ventricle mitochondria were isolated by differential centrifugation. NHE1 inhibition was obtainedusing 10 μM HOE. Data were expressed as mean ± SE. Statistical analysis was performed by one-way ANOVA followed by Tukey´stest. The experimental protocol was approved by the Animal Welfare Committee of La Plata School of Medicine. Results: Ourresults showed increased NHE1 expression in the mitochondria from ob-/- mice. Preliminary results also show increased NCLXexpression. These mitochondria also presented altered swelling, mPTP opening, CRC, and ΔΨm, while NHE1 blockade partiallyreverted this phenotype. Finally, mitochondria from ob-/- mice present reduced calcium content and increased NCLX expression.Conclusions: The role of mitochondrial NHE1 is not completely understood however, NHE1 activity could not directly result inΔΨm alteration considering its electroneutral exchange. However, alterations in mitochondrial Na+ concentration could influenceNCLX activity. Considering that NHE1 inhibition partially reverses mitochondrial alterations and that our preliminary data indicatesincreased NCLX, our results indicate that both mitochondrial NHE1 and NCLX are involved in the development of mitochondrialdysfunction in DCM.Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT), Fondo para la Investigación Científica y Tecnológica (FONCyT)Grant PICT 2017-1339 to CJDG.FD814JLMaternal DHA supplementation reverts the higher expression of proinflammatory genes and DNA methylation patterns in theoffspring ́s cord blood monocytes of women with Pregestational Obesity.Macarena Lépez1, Cristina Silva2, Bárbara Riquelme3,4, Cherie Hernández3,4, Karina Carrasco4, María Luisa Garmendia5, PaolaCasanello3,4(1) Pontificia Universidad Católica de Chile, PhD Program in Medical Sciences, Faculty of Medicine, Santiago, Chile.(2) Pontificia Universidad Católica de Chile, Biochemistry program, Faculty of Biological Sciences, Santiago, Chile.(3) Pontificia Universidad Católica de Chile, Department of Neonatology, Faculty of Medicine, Santiago, Chile.(4) Pontificia Universidad Católica de Chile, Department of Obstetrics, Faculty of Medicine, Santiago, Chile.(5) Universidad de Chile, Institute of Nutrition and Food, INTA, Santiago, Chile.The offspring of women with pregestational obesity (PGO) have an impaired innate immune function. Docosahexaenoic acid (DHA)supplementation during pregnancy decreases maternal systemic inflammation. Its effects on fetal immune response remainunknown. Aim: To determine the effect of DHA supplementation during pregnancy on gene expression, function, and DNAmethylation of PGO neonatal monocytes. Methods: Pregnant women with PGO (BMI ≥30 kg/m2) participating in the RCTNCT02574767, were supplemented with DHA: 200 mg/day (PGO-200, n=18) and 800 mg/day (PGO-800, n=21). Control group wasnormal-weight women (NW, n=20). At delivery, cord blood monocytes (CMo) were isolated to determine pro-inflammatory(MCP1, TNFα, IL-6, IL-8) and anti-inflammatory (PPARγ, PGC1α, IL-10) mRNA by RT-qPCR, and methylome analysis (EPIC-850K,Illumina®). NW and PGO CMo (n=8) were stimulated in vitro with Lipopolysaccharide (LPS), DHA, and DHA receptor antagonist,and then mRNA and protein levels were measured by RT-qPCR and CBA BD Biosciences, respectively. All subjects provided signedconsent. Study approved by the Institutional Ethics Review Board of PUC. Statistical: Kruskal-Wallis test, STATA. Results: PGO-200CMo expressed higher mRNA levels of MCP1, TNFα, IL-6, IL-8, PGC1α, and IL-10, but lower mRNA levels of PPARγ compared toNW. Pro-inflammatory changes and PPARγ levels were reverted in PGO-800 nearly to NW values. Hypermethylated sites werefound in PGO-800 compared to NW, mainly in the gene body and intergenic regions of genes related to immune response, andmetabolism. In vitro LPS stimulation induced CMo expression of MCP1, IL-6, and IL-10, in both groups and it was reversed by DHA.However, PGO-CMo had a blunted response to LPS challenge compared to NW. These effects were independent of the DHAreceptor (GPR120) activation. Conclusion: PGO induces the expression of inflammatory genes in CMo, with a blunted response toLPS. Maternal DHA supplementation reverts these changes and modulates DNA methylation in a DHA receptor-independentmanner.Fondecyt # 1171406 and # 1150878 and PIA-Anillo ACT172097.CB817BMStudy of the biochemical and behavioral effects of omega-3 fatty acids on normotensive and hypertensive ratsMaite Zavala2, Franco Dolcetti1, Romina Vazquez1, María Laura Fanani3, María José Bellini1, M. Celeste Villa-Abrille2, María LucreciaLongarzo1, Sabina M. Maté1(1) Instituto de Investigaciones Bioquímicas de La Plata (INIBIOLP); UNLP, La Plata, Argentina.(2) Centro de Investigaciones Cardiovasculares de La Plata (CIC); UNLP, La Plata, Argentina.(3) Centro de Investigaciones en Química Biológica de Córdoba, UNC, Córdoba, Argentina.Introduction: The dietary intake of polyunsaturated fatty acids (PUFAs) of the omega-3 series -eicosapentaenoic acid (EPA) anddocosahexaenoic acid (DHA)- reduces the risk of cardiovascular diseases (CVD) and produces beneficial effects in patients with